HUMAN ALPHA-GALACTOSIDASE-A - GLYCOSYLATION SITE-3 IS ESSENTIAL FOR ENZYME SOLUBILITY

Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the homodime ric glycoprotein that hydrolyses the terminal a-galactosyl moieties fr om glycolipids and glycoproteins. The type, site occupancy and functio n of the N-linked oligosaccharide chains on this lysosomal hydrolase w ere determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharid es respectively. When expressed in COS-1 cells, glycoforms with glycos ylation site 1 or 2 obliterated had more than 70 % of wild-type activi ty, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40 %); littl e, if any, was secreted. Expressed mutant glycoforms in which site 3 a nd site 1 or 2 were obliterated had little, if any, intracellular or s ecreted enzymic activity, and immunofluorescence microscopy revealed t hat the expressed mutant glycoforms were retained in the endoplasmic r eticulum, presumably where they were degraded. Thus glycosylation at s ite 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligos accharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzy me or mannose-terminated enzyme expressed in Autographa californica ce lls also aggregated when concentrated, emphasizing that site 3 occupan cy by a hybrid-type oligosaccharide was required for enzyme solubility .